The long-term goal of this research is to develop culture-independent strategies to effectively engineer and program the human microbiome in vivo. To this end, the objective of this proposal is to develop an enabling technology platform, named Genome Engineering by Self-Transmissible Replicons (GESTR), to allow the efficient delivery, propagation and expression of exogenous genes in the human microbiota, to characterize the kinetics of gene propagation in the microbiome, and to initially demonstrate the utility of such techniques in engineering the gut microbiota of mice. The specific goals of this work are i) to construct engineered self- transmitting conjugative transposons containing trackable payload genes and characterize their ability to mobilize into different strains typically found in the human gut microbiota; ii) to develop libraries of payload constructs to characterize transcriptional efficiency and codon adaptation for optimal payload gene expression in dominant gut microbes; and iii) to measure gene flow and transmission of exogenous genes by engineered self-mobilizable genetic elements in the gut microbiota of a murine model. Newly developed tools including Multiplex Automated Genome Engineering, de novo DNA-microarray-based gene synthesis, and meta- transcriptomics will be utilized to facilitate the development of this project. The proposed research will provide the first demonstration of key functionality and the development of sufficient new understanding to enable the broader use of this technology platform in future applications. Key questions in the dynamics of transmission and natural selection of laterally shared genes in the human microbiome will also be addressed. Engineering the human microbiome with augmented capabilities to report, prevent and reverse disease states and to modulate the metabolism of foods and drugs promises to be a critical avenue towards transforming human- associated microbes into micro-sensors, miniature protein-production factories, and adaptive bioremediation systems. This technology holds potential for development of clinical therapeutics of common microbial- associated diseases such as those of the gut (e.g. Crohn's, IBD, chronic maldigestion), oral cavity (e.g. dental caries), urogenital tract (e.g. infections, STDs), and skin (e.g. ectopic eczema).